Production of soy sauce

ABSTRACT

SOY SAUCE IS PRODUCED IN A RELATIVELY SHORT TIME BY FERMENTING MAROMI WITH AN INOCULANT SELECTED FROM SACCHAROMYCES AND DEBARYOMYCES OR A MAROMI ISOLATE.

United States Patent ABSTRACT OF TnE-nrscLosu E Soy sauce is produced ina relatively. short timeIby fer- Inenting Maromi with an, inocu lantselected ,ffrom Saccharomyces and Debaryomyees or a Maromi isolate.

The present invention relates to the production .of soy sauce and to soysauce having the'naturaltflavor and taste of aged soy sauce. Theinvention further relates to such soy sauce which may be produced intnr-elativelyshort time. I

Historical records indicate, that the Chinese have been using soybeanproducts. forwell over 3,000 years. Awicle yariety of foods havebeendeyeloped by the Chinese based on soybeans as a principalingredient. Soy sauce, one of the older Chinese products, has become animportant condiment in the United States,.not only for its use as. aflavoring'agent alone, but as an ingredient for mixing with otherflavoring agents to produce additional sauces and the like, e.g.Worcestershire sauce, which contain from 10 to soy sauce- Soy sauce maybe manufactured by two main processes. t v Soy sauce may bemade byfermentation or enzymati hydrolysis of protein to give amino acids andlow -molecular-weight polypeptides or by the acid hydrolysis process.The former of these two processes, i.e .t he fermentation process, is byfar the more important.proce ss,.since it gives the soy sauce a fullbody and flavor asopposed to the acid hydrolysis process. Hence, most ofthe soy sauce, especially the higher quality Oriental type soy sauce,

manufactured the United States is made by the fer.-

mentation process. a

Briefly stated, the fermentation process for preparing soy saucecomprises preparing a mixture of washed, cooked and drained soybeanswith parched wheat, or other grains or cereals, and adding to themixture (without addi- H tional water being used) a Koji culture. TheKoji culture is a naturally produced culture, and the exact compositionof :Koji culture is not fully known. However, generally speakingKojiculture containsmold, yeast and lacticacid bacteria. The mixture ofsoybeans, parched wheat and the days and this biologically alteredmixture is called Koji. The'Koji is added to an aqueousfsaltf'solu'tion. The' mixture of aqueoussalt solutio' i d Koji iscalled Maromi, The Ma'rorni is then allowe @fermentlfor an extended timeperiod. Whilefsome soy" sauce flavor can be developed after fermentationo the Marorni for as little as 30 days, for full-bodied andfull-flavored soy sauce, the fermentation'is'normally allowed to proceedfor at least six months and preferablyupto a y'earor more, efg. l8,mfonths. After the fermentation is complete and the'desired degree ofbody and flavor has been reached, the fermented mass is pressed and theliquid,obtainedf'therefrorn is soy sauce. The soy sauce may be furtherrnixed with otheragents such as salt, coloring, preservatives andthe'like. As can be ."y a i sirable to' short'enthe time of the Maromifermentation s'teprequired to produce a full-bodied and full-flavoredsoy sauce. Equally important,.however, is the requirement that-anyshortened fermentation timeof the Maromi must still'result in afull-flavored; and full-bodied soy sauce, .of the quality expected bythegeneral-consuming public.

' Therefore it is an object of this i'nvention toprovide a process. forpreparing a fullbodied and full-flavored soy sauce with a shortenedMaromi-fermentation time.,It is a further object of'the .inventionito'pr ovide a soy sauce of the fermented type which is;full-flavoredand-full- Ib0' di edj ;-but which has been-produced ifromiMaromi that was fermented in arelatively short period; of time. Otherobjects ofthis invention'will-be apparent from the follow- ,ingdisclosure and claims ;v i )5 1 Basicto the present invention isthedlscovery that certain organisms will develop, the .characteristie soysauce flavor in fermenting Maromi. Having. discovered these flavorproducing; organisms, a process has been devised wherein these organismsare grown during the fermentation of Maromi at ayery rapid rate to,.-prod uce the characteristic soysauce flavor a relatively shortfermentation time. 1" In order .to completely appreciate .thesignificance of the present invention, referencev isrnade in detail tothe vconventional.prg'icess.for producing a fermented soy sauce. Theconventional processis 'gener ally referred to, in the art as the Kojiand 'Mar'o'rni process. The'KojiIculture is essentially a starterinoculannf the total composition 'of which is not fully known to theart, as noted above. In a general sense, Kojiculture'is simplya'biolo'gically active portion of a Koji and which may be optionallyconce'n trated and dried. The origin of the first Kojicultureis lost inantiquity and inthe Koji and Maromi process it is necessary to have aKoji culture 'orderjto'preduce addi tional soy sau'ce, Koji cul'e'l'ca'n be obtained on the open market, and the principalfsources ofKoji; culture are Japan'and Chinafespec ially Japan. K jiculturemay beobtained in a liquid form "as well *as't dry form, noted above, and maybe mixed witliprepa'red'soybeans'and wheat to produce additional Kojiculture or produce Koji from which soy: sauce is made-by fermentation:In other words, Koji culture is a starter culture'fof not fully knowncomposition which has been Handed down from generation to generation foruse in produ g soy sauce via the 'Koji and Maremi process- 4 In thepreferred process in the art, an qual mixture of "roasted soybeansfa'ndboiled soybeans? (with excess liquid removed) is mixed withequal amouht of roasted wheat.-1f ter thorough mixing of the roasted andboiled :soyb'eans 'with the roasted Wheatythe'mixture isplaced in Kojiculture .is allo ed to biologically alferfor4 to 10 I Suitabl Vesselinomflatedwitb-Kofi cultl-lfe- Th Koji'culture may be concentrated'andpowdered Koji culture. 'whichis commercially available or the Kojiculture 'may be simply a biologically. active part of apreviously"produced.KojiprGenerally from -1 to 10% bywei'ght"ofiithezsoybean,'wheat; etc. of the Koji culture, which'isobtained from a previously produced .Koji, is used as the inoculant inthe mixture. 3, Whemcon'centr'ated powdered Kojicul-ture is used as the'inocu-lant, the amount thereof maygvarybetween 51 to 100 partsby'weight uofipowdered Koji;'-culture to 1,000 to noted from the abovediscussion of the fermentation process for producing soy sauce, thefermentation step requires that the Maromi'be allowed to ferment for anextended period of time and that large holding vessels for fermentingthe mixture are necessary to produce. sizable amounts of soy-sauce. Ascan be also appreciated, it would be detureproduces growth mainly. oflargely unidentifiedmolds,

yeasts and bacteria in the mixture of soybeans and wheat.

(This mixture of soybeans, wheat and growing Koji culture organisms isreferred to as Koji.) After this initial fer- ;mentation period, themixture of soybeans and wheat with the Koji culture organisms growingtherein (now referred to as Koji) is placed in an 18 w./v. percentaqueous salt solution. Generally, from 5 to 40 w./v. percent of the'Kojvi is placed in the salt solution. This mixture of the Koji. andsalt solution is referred to as Maromi. The Maromi-is mixed in a largetank-to obtain an even distribution. Thereafter mixing may .or' may notbe performed, depending upon the particular manufacturer making the soysauce. Mixing of the Maromi-is, however, not necessary. The Maromi isthen allowed to ferment at 60 to 110 F., especially 68 to 90 F. andideally 80 to 84 F. for an extended period of time. While this extendedperiod of :time may vary widely depending upon the flavor, body andbouquet desired, good quality soy sauce is only pro- (laced withfermentations which are allowed to proceed for; at least 9 months andthe better full-flavored soy sauce is allowed to ferment for. as long as18 months, especially the flavor associatedv with Oriental style soysauce. At'the end of the fermentation period, the liquid associated withthe Maromi is separated from the solids. The solids are discarded andthe liquid is further processed, e.g. pasteurized and compounded withanother ingredients to produce soy sauce.

Broadly stated, the present process for producing soy sauce comprisesfermenting conventional Koji, as described above,- in the presence ofspecific. organisms, as hereinafter defined, and in a salt solution withmixing under an inert gas blanket with short periods of, aeration,continuing the mixing and fermenting with intermittent aeration for upto Srweeks and pressing the resulting fermented mixture to obtain afull-flavored, full-bodied, Oriental style soy sauce.

It is greatly preferred that the Koji be comminuted to a relativelysmall particle size, e.g. by grinding. After grinding, all particlesshould pass through a number 3 U.S. sieve screen, but much improvedresults are obtained when all of the particles will pass through atleast a number 6 U5. sieve screen, and especially the average partidlesize should be less thana.l,000 microns, e.g. 1 to h 100 microns. Anyconventional type grinder or comminuting apparatus may be used forcommuting the Koji, for example, an Urschel mill, a geargrinder, aReitz. mill or a stone grinder. However, other grinders such as ballgrinders, rod grinders, hammer mills, etc., may be used. It is onlynecessary that the particle size afterlgrinding be within the rangenoted above. However, it izs-most important that during grinding thetemperature of the Koji be kept below 145 F., especially below 130 F.,e4g. below 100 F., in order to prevent the destruction of the enzymescontained in the Koji. Salt may be added to the Koji either before orafter grinding. Conveniently, the salt is added to the Koji prior togrinding so that the salt may be ground to a very small particle sizeduring the grinding process. Alternately, instead of grinding the Koji,the soybeans, wheat, etc., from which the Koji is made may be groundprior'to making the Koji, in the same manner as described above. In thiscase, the above noted temperature requiremerits are not so critical.

The Koji is then added to water and at this point salt is added, if thesalt was not added during the grinding process. The resulting mixture ofKoji, salt and water (called Maromi) should contain from 5 to 40 w./v.percent 7 of the Koji, especially between 20 and 25 w./v. percentrTheamount of salt in the resulting mixture, added either prior to grindingor after grinding, should be be tween about 6 to 30 w./v. percent,especially between 15' and 20 and ideally about 18 w./v. percent. Whilecommon sodium chloride is the preferred salt, especially in view of therelatively low cost thereof, other salts may be used, e.g. halides,especially the chlorides,--'df-.a-lkaline earth and alkali metals orammonia. In anyeventathe saltmust 4 J be an acid salt and particularlysuitable are sodium and potassium chloride, calcium chloride andmagnesium chloride; Organic salts may also be used, if desired, e.g.sodium acetate and sodium lactate. Nevertheless sodium chloride, is thepreferred salt and will normally be used in the process.

Themixture of Koji in the aqueous medium (hereafter referred'to asMaromi) is then agitated and to the agitated Maromi is introduced aninoculant, as hereinafter further defined.

After-inoculation, the Maromi is initially fermented for from 3 to 14days. Of course longer periods than 14 days may be used but there is nosubstantial advantage in continuing the fermentation longer than 14days, and generally no advantage is obtained in continuing thefermentation beyond 7 days. The initial fermentation temperature will bebetween 60 and 110 F. and especially between 68 and 90; F., eg. and 84F. The initial fermentation is carried out in a closed vessel having aninert gas head space. But preferably, at least some intermittentaeration is carried out during the initial fermentation. The inert gasprovides essentially anaerobic conditions and the anaerobic conditionsmay be insured by continuously or intermittently flushing with the inertgas. The intermittent aeration may be carried out by flushing the headspace of the vessel with an inert gas, such as nitrogen, containing lowlevels of oxygen. For the purposes of this specification and inert gasis defined as any gas which is nonreactive toward the Maromi.

Agitation during this initial fermentation period is not essential whenno aeration is performed, but agitation is required, at least duringpart of each aeration step, when aerationis used.

As noted above, during this initial fermentation period, the fermentingMaromi is preferably aerated for short periods of time. This may beaccomplished by stirring the fermenting Maromi and sweepingoxygen-containing gases, e.g. air, through the head space of the closedvessel. The amount of oxygen in the head space of the closed vesselduring these aerations may be quite low; similarly the dwell period tothe oxygen containing gas may be quite short, and can vary from asquickly as the oxygen containing gas can replace the inert gas in thehead space and the oxygen containing gas can be subsequently replaced bythe inert gas to up to 2 or 3 hours or more. It is not necessary thatthe oxygen containing gas be allowed to dwell in the headspace for anyextended period of time and only a momentary displacement of the inertgas in the head space is necessary. However, generally speaking, theoxygen-containing gas will be allowed to dwell in the head space from 1to 50 minutes, especially from 4 to 15 minutes and preferably about 5minutes. The frequency of aeration can vary widely and may be from aslittle as once every four days to as often as once every 10 minutes,especially from once every 2 days to 5 times a day and preferablyapproximately'Z to 3 times a day. Of course, during this aeration periodthe fermenting Maromi should be agitated.

The purpose of these intermittent aerationsteps is to essentiallyaccomplish in the fermenting Maromi a microaerophillic condition, i.e. astate of oxygen tension that is less than atmospheric and being on theborder between aerobic and anaerobic conditions. As can be appreciatedfrom the above, the initial fermentation can be under either anaerobicor microaerophillic conditions, but microaerophillic conditions aregreatly preferred. It has been found that microaerophillic conditions,at least for part of the fermentation time, develop a superiorfullfiavor and full-body soy sauce according to the present invention.

Alternately, instead of intermittent aerations, as discussed above,microaerophillic conditions may be established on a continuous basis byemitting with the inert gas an extremely low level of oxygen into thehead space of the closed vessel. The amount of oxygen introduced withthe inert gas into the head space of the closed vessel will vary widelydepending upon the volume of the head space, the volume of thefermenting mixture, the temperature of fermentation, the state offermentation, etc. Hence no specific amounts of oxygen can be stated asa general case, but one skilled in the art can readily determine whenmicroaerophillic conditions are produced within the fermenting Maromiand adjust the oxygen content to maintain those microaerophillicconditions. However, when the process is carried out using continuouslow levels of oxygen in the gases flushing the head space, it isnecessary to continually determine that microaerophillic conditions arebeing maintained in the fermentation process. This of course requires aconsiderable amount of effort in following the fermentation process andfor this reason it is preferred to intermittently aerate the fermentingMaromi, as noted above, rather than attempting to carry out thefermentation with continuous but low levels of oxygen in the inert gas.

After the initial fermentation period, preferably with aeration asdescribed above, the fermenting Maromi is further fermented for anadditional to 30 days or longer, if desired, either in a sealed vesselor with low levels of intermittent or continuous aeration. Hence, thesecond fermentation may be anaerobic, microaerophillic or mildlyaerobic, but microaerophillic or mildly aerobic conditions are greatlypreferred, since these conditions produce a superior ultimate fiavor.Preferably the Maromi is agitated during the aerations.

It should also be clearly understood that while the best flavor, bodyand bouquet are developed according to the present process when aerationis carried out in the second fermentation step, an acceptable, butinferior flavor, bouquet and body may also be obtained without anyaeration during the subsequent fermentation step and wherein thesubsequent fermentation step is carried out under an inert atmosphere.However, since superior fiavor, body and bouquet are developed when thefermentation is carried out with the above described aerations in thesecond fermentation step, this is the preferred embodiment of theinvention.

The second fermentation may be carried out at the same temperatures asthe initial fermentation and indeed the same procedure for introducingoxygen into the head space may be used. Hence, the second fermentationstep may be simply an extension of the first fermentation step,especially where microaerophillic conditions are used in both the firstand second fermentations.

After the subsequent second fermentation step, the solids are separatedfrom the liquid by any conventional means, e.g. filtering, pressing,centrifuging,- vacuum distillation, etc. The recovered liquid is thenmixed with conventional additives such as salts, edible coloring, e.g.caramel, other flavors, preservatives, sweeteners, such as sugars,spices, etc. to produce the finished soy sauce ready for sale to theconsuming public. Alternately, the product may be pasteurized eitherbefore or after the addition of the additives or before packaging. It isnot necessary, however, to pasteurize the soy sauce and indeed someadditional flavor will develop after packing the soy sauce when the soysauce has not been pasteurized.

The inoculant used with the present process is a yeast of the familyEndomycetaceae, subfamily Saccharomycetoideae, tribe Saccharomyceteae,and particularly useful yeast of the said tribe are selected fromSaccharornyces and Debaryomyces.

For example, species and varieties which may be used includeSaccharomyces cerevisiae, Sacclzaromyces cerevisz'ae var. ellipsoideus,Saccharornyces.pastorianus, Saccharomyces rouxii, Saccharornyces rouxiivar. polymorphus, Saccharomyces exiguus, Saccharornyces marxianus,Saccharornyces bailii, Saccharornyces logos, v. Laer et Denamur,Saccharornyces bayanus, Saccharornyces willz'anus, Saccharomyces uvarum,Saccharornyces delbrueckii, Saccharornyces delbrueckii, var. mongolicws,Saccharomyces carbergensis, Saccharornyces fragilis, Saccharomyceslactis, Saccharomyces rosei, Sacchromyces chevalieri, Saccharornycesbisporus, Sac'charomyces pastori, Saccharornyces fermentati,Saccharomyces hetero genicus, Saccharornyces microellipsodes',Saccharomyces oviformis, Saccharornyces mellis, Saccharornyces italicus,Saccharornyces florentinus, Saccharornyces acidifaciens, Saccharornycessteineri, Saccharornyces frizctuum, Saccharomyces elegans, andSaccharomyces veronae. All of the species and varieties are known to theart and are easily obtainable.

The particular species of Debaryomyces which may be used includeDebaryomyces hansenii, Debaryomyces kloeckeri, Debaryomyces subglobosus,Debaryomlyces vini, and Debaryomyces nicotianae. Likewise the speciesare known to the art and are readily obtainable.

For a complete discussion of the above-noted yeasts see The Yeasts,Lodder, 1., et al., North-Holland Publishing Company, Amsterdam, 1967,especially pages 117-122 and 270-279, which disclosure is incorporatedherein by reference. The foregoing reference gives the key toDebaryomyces and Saccharornyces for identification and isolationpurposes.

However, in a preferred embodiment of the invention the inoculant isisolated from Maromi since the isolated inoculant gives a better flavorto the soy sauce as opposed to the flavor obtained by using commerciallyavailable organisms. The isolated inoculant can be obtained by thefollowing procedure. Prepare two aqueous solutions of YM agar powder (acommercially available material and fully identified hereinafter), onewith 18 w./v. percent of NaCl and one with water only (approximately 5grams of YM agar powder per ml. of aqueous NaCl or water). Sterilize thesolutions, e.g. at 250 F. for 15 minutes, and prepare plates by placing15 to 20 cc. of, each solution in separate sterilized petri dishes andcool to l13l20 F. Inoculate each plate with approximately 0.1 to 0.3 cc.of one year old Maromi (produced by the conventional process as notedabove) by streaking out with a bacteriological needle, as is the commonpractice in the art.'Incubate the inoculated plates for about four daysat 71 F. (some growth will be noted after 48 hours). After incubation,with a bacteriological needle, remove the yeast growths from both thewater and saline plates (one skilled in the art can easily identifyyeast growths from bacteria and mold growths by visual observation) andplace each different yeast growth on a separate YM agar plate preparedor YM agar plate with NaCl, as discussed above. Of course, yeasts takenfrom YM agar plus salt plates are placed on new YM agar plus salt platesand yeast taken from YM agar plates are placed on new YM agar plates.Serially grow the yeasts on new YM agar plates and YM agar plus salt,respectively, as discussed above, and separate the yeast from each plateand grow on new :plates again until essentially pure cultures of yeastsare obtained and the cultures are essentially free from bacteria andmold cultures. Of course, the serial growing of the yeasts isaccomplished at the above temperatures and times. 'The number of serialgrowings of the yeasts will, of course, depend upon how carefully theyeasts are removed from the plates.

The above procedure is simply that for obtaining pure cultures by serialgrowths and is 'quite standard in the art and one skilled in the artwill have absolutely no trouble in obtaining pure yeast cultures.

After all of the yeast cultures have been purified, as discussed above,the purified cultures are visually observed under daylight and comparedwith respect to the following appearances: dull, shiny (glistening) andflaky. The observer will see that some of the yeast cultures arestrikingly different from other of the yeast cultures in these threerespects, and dull, shiny and flaky cultures will be readily apparent.(With proper streaking of the first Maromi culture, these threeappearances will be readily apparent in the first culture of the Maromiand by retaining only the cultures having these three appearances thenumber of cultures and number of serial growing may be materiallyreduced.)

The cultures having a dull, shiny or flaky appearance will be thosecultures containing the organisms of the present invention. Any one ofthese cultures may be used to produce soy sauce, but a combination ofthe three cultures produce an exceptional fiavor, body and bouquet.

An alternate procedure for obtaining the Maromi isolated organisms maybe used. The procedure is as follows. Into distilled water is placed 4w./v. percent of agar and cooked at boiling to dissolve the agar. Thedissolved agar solution is autoclaved and cooled to room temperature, orat least below 45 C. Several plates are made from the agar by mixing theagar with an equal volume of naturally produced soy sauce (produced bythe conventional Koji and Maromi process). The soy sauce used in mixingwith the agar should be essentially pure soy sauce and suitable purityis obtained by passing the soy sauce through a bacteriological filter.After the mixture of soy sauce in agar solution has solidified in theplates, the plates are inoculated with aged Maromi obtained from aconventional Koji and Maromi process by streaking the plates with abacteriological needle. The Maromi used for streaking the plates shouldbe at least one month old Maromi but Maromi of twelve months old orlonger is desired. The plates are then placed in a conventionalanaerobic jar or any other like device to obtain anaerobic conditions.The plates are incubated at mild temperatures, e.g. up to 40 0,especially at room temperature, for 2 to 4 months under anaerobicconditions. After about one month and especially after about two months,distinct colonies of organisms will be observed on the plates. Therewill be three very distinct and easily recognizable dilferent coloniesand essentially no other colonies of any appreciable size will becontained on the plates. These three distinct colonies, by visualobservation, will be characterized in that one will be dry, flaky,white, large and rough; the second will be large, creamy (butteryconsistency); and the third will be mucoid and having a butteryconsistency. If any doubt remains to the observer, although withreasonable care in producing the plates there should be no doubts, thecolonies can be further identifled by microscopic examination. Thefirst-named colony will have organisms of 6 to 10 microns, thesecond-named colony will have organisms of 6 to 10 microns, and thethird-named colony will have organisms of 2 to 4 microns. The threedistinct colonies noted above, are picked from the plates and placed onseparate plates made of YM agar with 18 w./v. percent of NaCl and thecolonies are further grown to increase the amount thereof for at least24 hours at mild temperatures, e.g. room temperature, but the growth maybe continued-as long as desired. However, little additional growth takesplace after two weeks. The procedure of growing the separate colonies onseparate YM plates is repeated until a substantial amount of theseparate colonies is obtained. While no minimum amount is required, itis more convenient to work at least A gram of each of the colonies andtherefore it is convenient that the growing of colonies on the YM platesbe repeated until gram of each of the colonies is obtained. Thisprocedure also ensures that the colonies have been purified, which is astandard bacteriological technique.

The separate colonies are picked from the YM agar plate with abacteriological needle and inoculated into tubes of yeast medium broth.Yeast medium broth is defined according to the standards of NorthernRegional Research Lab (NRRL). The yeast medium broth consists of 3 gramsof yeast extract, 3 grams of malt extract, 5 grams of beef extract, 10grams of glucose and 1,000 cc. of water i.e. essentially YM powderwithout the agar. The broth is prepared by boiling to dissolve andautoclaving to sterilize. One-third of the broth is placed in a flask.To this one-third ofbroth is added 1 w./v. percent of Tween (Sorbitanmono-oleate) to lower the surface tension thereof. Salt (NaCl) is addedto the Tween 80 containing broth and to additional flasks of the brothwithout Tween 80 to produce an 18 w./v. percent solution of the salt.One flask is inoculated with the first-named organism (the dry, flaky,white, large organisms); one flask is inoculated with the second-namedorganism (large, creamy, buttery consistency organisms); and one flaskis inoculated with the third-named mucoid organism. The first andsecond-named organisms are inoculated onto flasks which do not containthe Tween 80 and the thirdnamed organism is inoculated onto the flaskwhich does contain Tween 80. All of the flasks are incubatedatmildtemperatures, e.g. room temperature for at least two weeks. Longer timesmay be used as desired, but little growth take place after fo'ur months.The growth is then separated from the medium of the flasks in anydesired manner, such as centrifuging (where the sediment of thecentrifuging is the organisms) and the separated organisms are thencombined and are suitable for use in inoculating the Koji in the mannernoted above.

For purpose of the present specification and claims, the organisms whichare isolated as described above by either technique, are defined by theterms Maromi isolated organisms and these terms are meant to describethe above noted isolated organisms. These terms, as used in the claims,are intended to convey this definition. When pure organisms, obtainedcommercially, are used, as opposed to isolating the organisms, as notedabove, it is also preferred that at least one Saccharomyces and at leastone Debaryomyces be used in combination to inoculate the Maromi,although only one of the two will produce an acceptable soy sauce. Thepreferred species are Saccharomyces rouxii and Debaryomyces hansenii. Inany case, at least 10 organisms per cc. of either the commerciallyavailable or isolated organisms must be used to inoculate the Maromi,preferably at least organisms per cc., and especially at least 1,000,e.g. 10,000 organisms per cc. of total liquid in preparing the Maromi,as noted above.

While the present invention has been illustrated in terms of producingsoy sauce of Koji made from soybeans and wheat, as is apparent to thoseskilled in the art, Koji may be produced with a combination of soybeansand other grains or cereals. For example, instead of wheat, there may beused other grains or cereals such as rice, barley, sorghum, cotton seed,corn or maize, oats or legumes uch as legume seeds. Generally theproportion of soybeans, on a weight basis, to other grains or cerealswill be between 25% to 75% soybeans and the remainder being other of thecereals or grains noted above. Preferably, however, the proportion ofsoybeans to other cereals or grains is approximately 50% by weight.Also, in the preferred process, one-half of the soybeans are roasted andone-half of the soybeans are boiled. The roasted and boiled soybeans arecombined with a cereal or grain in approximately equal proportion, e.g.with wheat (roasted). In addition, part of the soybeans may be replacedby other high protein content beans, such as peas, lima beans,black-eyed peas, kidney beans, etc. However, these other beans shouldnot replace more than one-half of the soybeans.

Hence, the term Koji as used in the present specification and claimsembraces the above noted variation of beans, cereals and grains. Thefollowing examples will illustrate the preferred embodiments of theprocess, but it should be understood that the invention is not limitedto the following illustrative examples but is applicable to the extentof the foregoing disclosure.

EXAMPLE 1 A conventional Koji was prepared by boiling in water at 212 F.for 15 minutes 8% pounds of soybeans, roasting for 1 hour in a rotatingoven at 250 F. 8% pounds of soybeans and roasting for 1 hour in arotating oven at 250 F. 17 /2 pounds of wheat. The boiled soybeans,EXAMPLE 6 roasted soybeans and wheat were mixed until a uniform mixturewas obtained. The mixture was ground with Urschel mill until allparticles had a size of less than 10 microns. To the resulting groundmixture was then added 0.1 gram of dry powdered Koji culture and allowedto ferment 5 days at 82 F.

This example illustrates the use of organisms isolated from naturalMaromi.

Five grams of YM powder* were added to 100 cc. of 18 w./v. percentsaline solution and sterilized at 250 F. for 15 seconds and designatedmedia 1.

Five grams of YM powder were added to 100 cc. of

EXAMPLE 2 water, sterilized at 250 F. for 15 seconds and designatedmedia 2. The Koji of Example 1 was l d i 167 pounds f Media 1 and media2 were cooled to 115 F. and 20 sterilized 18 w./v. percent aqueous NaClcontained in a Cc.. of each med Was Placed in Separate 3-inch diameterstainless steel mixing vessel fitted with a paddle stirrer andsteriliZed Petri dishes (designated dish 1 and dish mixed until a slurrywas d d, with mixing, 13 f Media 1 and 2 were streaked with l-year-oldnaturally inoculant in 18 w./v. percent saline solution was addedProduced Maromi and allowed to incubate at 71 F- f to the slurry. Theinoculant contained approximately y 1X10 organisms of Saccharomyces mii, The mixing With bacteriological needles, the yeast cultures(visually vessel was capped with a head plate having a nitrogen inletifie Which Were either flaky, dull or shiny were and a itrogen Outlet.Nitrogen was flushed through the removed from dishes 1 and 2 andstreaked in separate head space of approximately 2 cu. ft. at a rate ofapproxi- Petri dishes Containing the same, respective, media as mately10 cc./min. The slurry was maintained at between 20 scribed above. Thesedishes were incubated at 71 F. for 80 and 84 F.by means of a Waterjacket surrounding the 4 y This p y g procedure was repeated 3 times.mixing vessel. The above conditions were maintained for after W h p e Ct s f flaky, dull and shiny Y ast a total of 5 days, with a 15 minutestirring every 4 h cultures were obtained. The cultures were identifiedas Saccharomyces and Debaryomyces.

Equal portions of the flaky, dull and shiny cultures were After the5-day period, the nitrogen flush was stopped every 4 hours and replacedby an air flush for 15 minutes,

with stirring during the flush and with stirring for 15 used in the pm0f IXaIIlP1e ill the same total minutes after the air flush wascompleted and nitrogen amounts as 1n. Example 2, instead of theSaccharomyces was again flushed through the head space. After each aerauii- Th p o c d Soy sa ce had anexcellent flavor tion, the nitrogen flushwas resumed and continuously and full body.

maintained. The above temperature conditions, nitrogen EXAMPLES and airflushing schedule'was continued for a total of 21 days beyond theinitial S-day fermentation period (e.g., The pr'ofiedure of Exalhple 2Was pe except that m} of 26 d processing i the conditions and materials,as shown in Table I, were Thereafter, the liquid was separated from thesolids usede results are noted in the tableby means of a conventionalfilter press. The liquid had the color and aroma of soy sauce. The tastewas that of EXAMPLE 12 a good soy sauce with medium body. The soy saucewas The Ptoeethlfe 0f EXample 2 TePeated eXCePt that aged for two weeksin a stainless steel closed vat and the 110 aefatlofl Was la tduflhglthe P The fiavor developed into a very full-bodied good tastingsoy 4 sulting flavor was inferior to the flavor of Example 2, but

sauce was still the characteristic soy sauce flavor.

EXAMPLE 3 As can be seen from the foregoing disclosure, soy sauce can beproduced with very short total fermentation times, The procedure ofExample 2 was repeated except that e.g. in less than 2 months andespecially in about 30 days. the inoculant contained Debaryomyceshansenii. The re- This, of course, results in a significant saving intime and sulting soy sauce had a slightly better flavor than that ofmoney over the conventional periods of 1 year to 18 Example 2. months.

TABLE I First Second termental fermentation Example Koji inoculant temp.and time temp. and time Flavor 2 lbs. roasted soybeans 7 3 lbs. boiledsoybeans 1x10 Saccharomucea rauxiL.-. 70 F., 7 days.-- 80 F., 14 days"Good flavormediurn body.

30 lbs. roasted wheat 8 Same as above 1X10 Saccharomyces roux-ii do doMild flavor-light body. 9 d 1X10 rtmzii-. 83 F., 6 days". 83 F., 21 daysVery distinct flavor and full body.

20 lbs. boiled soybeans 10 5 lbs. roasted soybeans 1x10 Saccharomycesr0uzii 84 F., 7 days.-. 84 F., 25 days.. Strong flavor-full body.

12 lbs. roasted wheat 11 Same as above... do 105 F., 7 days 105 F., 21days Strong flavor, but slightly acid taste full body.

EXAMPLE 4 We claim:

1. In a Koji and Maromi process for the production of h Procedure ofEXamPIe 2 was repeated except that soy sauce, the improvement comprisinginoculating the the lhoculaht contained approximately equal amounts ofMaromi with an inoculant having as the sole essential f y a Debaryomyceshlmsenit- The organisms of said inoculant at least one yeast selectedl'esulhhg flavor was slghlficahtly Superior t0 that of either from thegroup consisting essentially of Saccharomyces, EXamP1eZOIEXamPIe3-Debaryomyces, Maromi isolated yeasts and mixtures EXAMPLE 5 thereof,said inoculated Maromi having at least 10 yeast The procedure ofExamples 2, 3 and 4 were repeated except that the air flush used inthose examples after the powder is YM agar (dehydrated) as disclosed infirst 5-day period was instead also used during the first Wiekerham.Tech Bull- 1029. DA. 1951 and contains S-day Period. In each case abetter flavor was obtained. Z53 g gggf afif gggg fg g gii agar- This isa Stand organisms per cc. of liquid initially fermenting said inoculatedMaromi in. a closed vessel having an inert gas head space for at leastabout 3 days at a temperature between about 60 and 110 F. underanaerobic or microaerophillic conditions and subsequently furtherfermenting for at least days at a temperature between 60 and 110 F.under anaerobic, microaerophiillic or mildly aerobic conditions andseparating the liquid of the fer: mented Maromi from the solids, andwherein the total fermentation time is no more than about 5 weeks.

2. The process of claim 1 wherein the said Maromi is produced from aKoji which was ground to a particle size which will pass through a No. 3US. sieve screen.

3. The process of claim 2 wherein the particles will pass through a No.6 US. sieve screen.

4. The process of claim 3 wherein the said Koji was I ground at atemperature below 145 F. and has an average particle size of less than1,000 microns.

5..The process of claim 4 wherein the said temperature is below 130 F.

6. The process of claim 1 wherein the said Marorni contains an ediblesalt solution and the amount of salt in said solution is from 5-30 w./v.percent. 7

7. The process of claim 6 wherein the said salt is NaCl.

8. The process of claim 6 wherein the said solution contains from 15-20w./v. percent of NaCl.

9. The process of claim 8 wherein the said solution containsapproximately 18 w./v. percent of NaCl.

10. The process of claim 1 wherein Maromi is produced from Kojicontaining a grain or cereal.

11. The process of claim 10 wherein the grain or cereal is wheat.

12. The processof claim 11 wherein the said Koji contains from 25-75% byweight of soybeans and from 75-25% by weight of wheat.

13. The process of claim 1 wherein the Maromi contains at least 1,000yeast organisms per cc. of liquid.

14. The process of claim 1 wherein the yeast are selected fromSaccharomyces rouxii and Debaryomyces hansenii.

15. The process of claim 1 wherein the said yeast are Maromi isolatedyeasts.

16. The process of claim 1 wherein at least intermittent aeration iscarried out during said initial fermentation.

17. The process of claim 16 wherein the Maromi is agitated during saidat least intermittent aeration.

18. The process of claim '16 wherein microaerophillic 12 conditions areestablished for at least part of the said initial fermentation.

19. The process of claim 1 wherein at least intermittent aeration iscarried out during the said subsequent fermentation.

20. The process of claim 19 wherein the said aeration of the saidsubsequent fermentation is carried out with agitation.

21. The process of claim 19 wherein microaerophillic conditions areestablished for at least part of said subsequent fermentation.

22. The process of claim 19 wherein the aeration is intermittent, andthe time period of aerati onis less than 2 hours and the aeration iscarried out at least once" a day. g

23. The process ofclaim1 wherein the total fermentation time is 30 daysor less.

24. The process of claim 1 wherein the said yeast are Saccharomyces.

25.. The process of claim 1 wherein the said yeast are Debaryomyces.

26. The process of claim 18 wherein microaerophillic conditions aremaintained throughout said initial fermentation.

27. The process of claim 21 wherein microaerophillic conditions aremaintained throughout said subsequent fermentation.

28. The process of claim 1 wherein the initial fermentation is up to 14days and the subsequent fermentation is up to 30 days.

References Cited UNITED STATES PATENTS 3,495,991 2/1970 Mogi et a1.-99145 1,394,236 10/1921 Togano 99145 FOREIGN PATENTS 3,542 6/1962 Japan99145 139,609 3/1948 Australia 99-145 OTHER REFERENCES Hesseltine:Industrial Mycology, Shoyu Mycologia, vol. 57, 1965, pp. 174-177.

Lodder et al.: The Yeasts, North-Holland Pub. Co., Amsterdam,Interscience, N.Y., 1952, pp. 142-145.

A. LOUIS MONOCELL, Primary Examiner J. M. HUNTER, Assistant Examiner

